Production of xylose (dextrose) isomerase enzyme preparations

ABSTRACT

PROCESS FOR THE PRODUCTION OF XYLOSE (DEXTROSE) ISOMERASE BY MEANS OF A MUTANT STRAIN OF STREPTOMYCES THAT PROLEFERATES IN A CULTURE MEDIUM THAT MAY BE FREE OF XYLOSE. THE XYLOSE (DEXTROSE) ISOMERASE ENZYMES IS THEREFORE CONSTITUTIVE RATHER THAN INDUCED.

United States Patent ABSTRACT OF THE DISCLOSURE Process for theproduction of xylose (dextrose) isomerase by means of a mutant strain ofStreptomyces that proliferates in a culture medium that may be free ofxylose. The xylose (dextrose) isomerase enzyme is therefore constitutiverather than induced.

FIELD OF THE INVENTION This invention relates generally to enzymaticisomerization. More particularly, the invention relates to improvementsin the production of enzyme preparations that are useful for theisomerization of dextrose to levulose, and to their use for suchisomerizations. The invention also relates to novel isomerase enzymepreparations.

BACKGROUND The production of corn syrup and corn syrup solids, by thehydrolysis of starch, has progressed in the direction of ever sweeterproducts. The acid hydrolyzates that were the initial commercialproducts have gradually given way to generally superior productsproduced by the use of sacchrifying enzymes. The art has advanced to thepoint where enzyme hydrolyzates can be routinely produced on acommercial scale at D.E. values in excess of 95.

However, the industry is interested in even greater sweetness, andresearch has been conducted for years into techniques for isomerizingstarch hydrolyzates to increase the content of levulose. Importantinitial work in the field was conducted by Cantor and Hobbs, as reportedin US. Pat. 2,354,664, granted Aug. 11, 1944. Cantor and Hobbs utilizedalkali catalysis to effect isomerization.

Alkali isomerization is limited in the degree to which the isomerizationcan be carried out efiiciently and with the formation of commerciallyacceptable products. Consequently, there was a widespread search formany years for an enzyme that would effect the isomerization. Thissearch culminated in the discovery that xylose isomerase, whichcatalyzes the interconversion of D-xylose and D- xylulose, did convertD-glucose (dextrose) to D-fructose (levulose), as described by Marshalland Kooi in Science, Apr. 5, 1957, Vol. 125, No. 3249, pages 648- 649,and in the pioneer patent in the field, US. 2,950,- 228, to Richard 0.Marshall, on Aug. 23, 1960. Since that time, there has been a greatamount of research activity in connection with enzymatic isomerization.

The use of a microorganism of the Actinomycetales order, for theproduction of an isomerizing enzyme, was reported by Sato and Tsumura intheir paper, A Study on Isomerization of Dextrose by a StreptomycesStrain, at the Annaul Meeting of the Agricultural Chemical Society ofJapan held at Sapporo in July 1964. A great deal of subsequent work,relating to the use of microorganisms of the Streptomyces genus forisomerase enzymeproduction, has been conducted at the FermentationResearch Institute of Japan, as reported by Dr. Y. Takasaki and hisassociates. Some of this work has been summarized in the publication,Fermentation Advances, Academic Press, New York, 1969, in the article byDr. Takasaki et a1. beginning at page 561.

Patented May 28, 1974 The work of Sata and Tsumura led to the use ofStreptomyces microorganisms for the production of isomerizing enzymes bythe use of a nutrient medium containing xylose. Unfortunately, if xyloseis required for enzyme production, there are limitations on the natureand cost of the medium that is required.' Dr. Takasaki and hisassociates identified certain strains of Streptomyces that secretedxylanase, and that therefore could be cultured in nutrient mediacontaining xylan, which is much less expensive than xylose.Unfortunately, the economics and other limitations on the nature andcost of the culture media required even for these microorganisms imposesevere limitations on the process. Moreover, all known strains have beenthought to require the presence of cobalt in the culture medium forpractical enzyme production, and this created a disposal problem.

OBJECTS OF THE INVENTION One object of the present invention is toprovide new and improved practical techniques for the production ofisomerizing enzyme preparations that do not require the use of xylose toinduce the production of the enzyme.

A closely related object of the invention is to provide practicaltechniques for the production of isomerizing enzyme that do not requirethe use of cobalt in the culture medium.

A more general object of the invention is to provide practical processesfor the production of isomerizing enzyme for converting starchhydrolyzates or dextrose solutions to levulose-bearing products, thatare more attractive for commercial exploitation than prior artprocesses.

A related general object of the invention is to provide an improvedpractical process for the production of levulose-bearing products.

Other objects of the invention will be apparent hereinafter from thespecification and from the recitals of the appended claims.

DEFIKNI'IIONS Because of the plethora of terms that are in common use inthe art, a few definitions are made to simplify the present applicationand permit it to be more concise.

The term D.E. is an abbreviation for dextrose equivalent, and theseterms are used interchangeably to refer to the reducing sugar content ofa material calculated as dextrose and expressed as percent of totalsolids.

Starch hydrolyzate The term starch hydrolyzate is used in a general wayto refer to a syrup or dry product that is made by the hydrolysis ofstarch. Such a product may be made by acid or enzymatic hydrolysis, orby a combination of acid and enzymatic hydrolysis. A preferred type ofstarch hydrolyzate for use for isomerization in accordance with thepresent invention is produced by acid or enzyme thinning to a D.E. of 10or less, followed by enzymatic saccharification to a D.E. above 95, andpreferably above 97.5.

Glucose and dextrose Medium D.E. starch hydrolyzates are commonlyreferred to in the art as glucose, whether the starch hydrolyzate is inthe form of a syrup or in the form of solids. The term dextrose iscommonly reserved for the refined crystalline monosaccharide that isrecovered from a high D.E. starch hydrolyzate, or for D-glucose as aconstituent of starch hydrolyzates. As used hereafter, the term dextrosewill be used to embrace this monosaccharide inany form, in solution ordry, as a constituent of a starch hydrolyzate syrup, syrup solids, or inrefined crystalline form.

Fructose and levulose The terms fructose and levulose are generallyemployed interchangeably in the art to refer to the isomer of dextrosethat is sweeter than dextrose. This isomer is found in honey and ininvert sugar, along with dextrose, and it is valuable because of itssweetness. The term levulose will be used to refer to thismonosaccharide.

The enzyme The enzyme that isomerizes dextrose to levulose has beenreferred to in the art by several names. It is referred to in theMarshall patent, 2,950,228, as xylose isomerase, because it isomerizesxylose to xylulose. This activity is in addition to its ability toisomerize dextrose to levulose. It has also been referred to in the artas dextrose isomerase and glucose isomerase. The term xylose isomerasewill be used herein for reasons to be described presently, under theheading, Characterization of the Enzyme.

Enzyme preparation The term enzyme preparation is used to refer to anycomposition of matter that exhibits the desired xylose isomeraseenzymatic activity. The term is used to refer, for example, to livewhole cells, dried cells, cell extracts, and refined and concentratedpreparations derived from the cells. Enzyme preparations may be eitherin dry or liquid form.

Units In this application, all parts and percentages are by weight, andon an as is basis, unless expressly stated to be otherwise.

Isomerase unit One isomerase unit is defined as the amount of enzymeactivity that is required to produce one micromole of levulose perminute under the isomerization conditions described hereafter under theheating, Assay of isomerase Activity.

Streptomyoes This term refers to a genus of microorganisms of the orderof Actinomycetales. These microorganisms are aerial mycelium-producingactinomycetes. The genus is well recognized. Some of its importantdistinguishing characteristics are described, for example, in the text,The Actinomycetes, by Selman A. Waksm'an, The Ronald Press Company, NewYork, 1967, page 135 et seq.

BRIEF SUMMARY OF THE INVENTION We have now discovered that it ispossible to produce xylose isomerase enzyme preparations on a practicalbasis by cultivating in a nutrient medium a microorganism of the genusStreptomyces that is characterized by its capacity to form anappreciable quantity of xylose isomerase when cultivated in a nutrientmedium that is free from xylose and xylose-supplying material and thatis essentially free of cobalt.

The preferred microorganisms are mutant strains of Streptomyoesolivochromogenes, especially, S. olivo chro mogenes ATCC Nos. 21,713,21,714, 21,715, and their equivalents. These microorganisms have therequisite functional characteristics, that is, they do form appreciablequantities of xylose isomerase when cultivated in nutrient media thatare free of xylose and xylose-supplying material and that are free ofadded cobalt. While these specific microorganisms are preferred, it isbelieved that any microorganism of the genus Streptomyoes can be mutatedto secure a strain having the desired characteristics.

While it is possible, in accordance with the invention, to producesuitable enzyme preparations on a practical scale by cultivation of theselected microorganism in a nutrient medium that is free from xylose andxylosesupplying materials and that also essentially free of cobalt, evenbetter and more economical results are generally obtained when xylose,cobalt, or both, are present in the nutrient medium in which thepreferred microorganisms are cultivated.

DETAILED DESCRIPTION OF THE INVENTION Production of mutant strains Aparent strain, S. olz'vochromogenes ATCC 21,114, was selected formutation because it was known to be a good source of xylose isomerase.

This microorganism was exposed in the spore state to a dose ofultraviolet light sufficient to kill 97% of the exposed microorganisms.The irradiated spores were plated out, and each colony was tested forenzyme activity. Since the medium on which the colonies were growncontained no xylose to induce the formation of isomerase enzyme, onlythat colony that had the desired character, istics showed a positiveenzyme reaction. This colony was isolated and was extensively tested, todemonstrate that the new, mutant strain did indeed produce appreciablequantities of xylose isomerase without the need for xylose in thenutrient medium as in inducing agent.

In greater detail, the steps involved in this procedure were as follows.S. olivochromogenes ATCC 21,114 was grown on Difco starch agar, made to2.0% agar with added agar, until the culture had sporulated abundantly.These spores were then harvested and suspended in 20 ml. of a 0.1%solution of the surfactant, Tween (the trademark of Atlas Powder Co. fora polyoxyalkylene derivative of sorbitan monooleate). Then 0.1% of adispersing agent, Marasperse C (the trademark of Marathon Paper MillsCo. for a lignin-sulfonic acid dispersing agent) was added, and thesuspension was sonicated for two bursts of 15 seconds each, to break upchains and clumps of the spores. The resulting spore suspension wasexamined and was found to be relatively free of spore chains.

This spore suspension was exposed in a shallow dish, with stirring, toultraviolet light from a Westinghouse Sterilamp (782H-10) at a distanceof four inches for about eight minutes. This exposure produced a 97%kill of the spores. The suspension was then diluted, and at the properdilution to give about 100 colonies per plate, was streak out in Petridishes containing the following medium:

TABLE l.-AGAR MEDIUM pH to 7.5 with NaOH.

About 40 to 80 colonies per plate did grow and were subsequentlyisolated. These isolates were screened for the ability to isomerizexylose after being grown on a medium that did not contain xylose or anymaterial supplying xylose.

Isolation of and fermentation with the mutant I One isolate was foundthat produced isomerase activity when grown in the absence of xylose.The isomerase activity produced was 0.155 units/ml. After a secondtransfer, the activity was increased to 0.6 units/ml.

The culture was then carried through several more transfers and then wasplated out for reisolation. Eleven colonies were selected, grown uponslants, and then grown up through two seed stages. The eleven secondseed stages were used to inoculate two culture media, identified belowrespectively as Medium A and Medium B. In addition, for comparativepurposes, a seed grown from the parent culture, olivochromogenes ATCC21,114, was also used to inculate the same two culture media.

The procedure employed and observations made are described in detailbelow.

For the two seed stages, the medium employed had the composition setforth in Table 2.

TABLE 2.FIRST AND SECOND SEED MEDIUM pH to 7.0 with NaOH, with 1 drop ofan antifoaming agent per flask.

The first stage seed was carried out for two days, with 100 ml. of theseed medium in each of the required number of 500 ml. Erlenmeyer flasks,utilizing a reciprocating shaker at 28 C. Spores from each of therespective slants were used to inoculate each of these flasks.

The second seed stage was carried out for one day, with 200 m1. of theseed medium in each of the necessary l-liter Hinton flasks, using a Gumprotary shaker at 28 C. Ten-ml. portions of the first seed were used toinoculate each of the second seed flasks, respectively.

Five-ml. portions of material from the second seed stages were then usedto inoculate culture media having the following compositionsrespectively:

TABLE 3.-CULTURE MEDIA A AND B pH to 7.0 with NaOH.

For the fermentation, 100 ml. of the appropriate respective culturemedium was placed in each of the necessary l-liter Hinton flasks. Onedrop of antifoaming material was added to each flask and the flasks weresterilized, then cooled. After inoculation, the flasks were then placedon a Gump rotary shaker and maintained at 28 C. for two days.

Harvesting of the enzyme preparation After fermentation, the contents ofeach flask were centrifuged at 10,000 times gravity for 15 minutes. Thecell pack was then separated, weighed, and frozen for storage.

For assay or use, the cell pack or a proportionate part of it wasbrought back to its original volume with distilled water, and the cellswere resuspended. When reconstituted, the cell suspension was assayed inthe manner described below.

Conversion of the cell suspension to solubilized form To prepare theenzyme for assay, it is first necessary to convert it to a soluble form.A suitable means for accomplishing this is by sonication.

Cells from a known volume of culture broth are resuspended in 0.05 molarphosphate buffer (pH 7.5). The suspension is then sonified using aBranson Sonifier Model 185- D (20 kc.) until the microbial cells of thesame are sufliciently disrupted so that the isomerase enzyme issubstantially all liberated. Holding the sample tube in an ice bathduring sonication prevents overheating and enzyme inactivation.

6 The resulting enzyme preparation was a solution of solubilizedisomerase.

Assay of isomerase activity The assay procedure involved making aspectrophotometric determination of the ketose produced from a glucosesolution under a standardized set of conditions.

A stock solution was made up in the following manner:

TABLE 4.-STOCK SOLUTION FOR ASSAY Component: Amount 0.1 M ml..... 1 0.01M COCl2'6H2O ....lnl-- 1 1 M phosphate buffer, pH 7.5 ml 0.5 AnhydrousD-glucose g 1.44 Distilled water to make up a total volume of The enzymepreparation to be assayed was first diluted to contain from 1 to 6isomerase units per ml.

An enzymatic isomerization was conducted by adding 1 ml. of the enzymepreparation to 3 ml. of the stock solution, and incubating for 30minutes at 60 C. At the end of the incubation period, a 1 ml. aliquotwas taken and quenched in a 9 ml. volume of 0.5 N perchloric acid. Thequenched aliquot was then diluted to a total volume of 250 ml. As acontrol, for comparative purposes, a glucose blank was also run bysubstituting 1 ml. of water for the 1 ml. of the enzyme preparation insolution form, at the beginning of the incubation period.

The ketose was then determined by a cysteine-sulfuric acid method. Forthe purposes of this assay, one isomerase unit is defined as the amountof enzyme activity that is required to produce one micromole of levuloseper minute under the isomerization conditions described. The assayresults are summarized below in Table 5.

TABLE 5.ASSAY RESULTS Medium A Medium B (no xylose) (xyloso) Isom-Isomerase Dry cell erase Dry cell activity, weight activity, weight,Culture u./ml. gJliter u./ml. gJliter Parent strain 0.24 6. 45 7. 46 8.75 D 0. 04 6. 56 0. 63 9. 32

Mutant strains CPC 3, CPC 4, and CPC 8 were selected for continuedmaintenance. One week after securing the results reported above, each ofthese three mutants was again used to inoculate the two respectiveculture media, with the following results.

TABLE 6.SECOND ASSAY RESULTS These observations were considered toestablish that the mutants had the capability of producing xyloseisomerase without the need for xylose in the culture medium. Inaddition, it was observed that some of the cultures producedsubstantially more enzyme than the parent strain, when grown in aculture medium containing xylose. This characteristic is veryimportant'for commercial purposes, since greater productivity means thatless fermentor capacity is required for a given amount of enzymeproduction.

Eifect of the carbohydrate source in the culture medium To demonstratethe efiect of the source of carbohydrate in the culture medium, and alsoto demonstrate the ability of mutant strain CPC 3 to produce enzyme whencultured in the presence of a variety of carbohydrates as the solecarbon source, several selected strains of Streptomyces were cultured.The culture medium was identical in each case, except for thecarbohydrate present. Other conditions were held constant.

The observations made are recorded below in Table 7.

TABLE 7.EFFECT OF TYPE OF CARBOHYDRATE PRESENT ON PRODUCTION OFISOMERASE WITH STREPTOMYCES CULTURES D.E. starch Hydroly- GlyoerinMannose Glucose Hydrolyzate 1% 2% 2% 1.5% zate 2% xylose 2 a Glucoselsomerase activity, units/ml. Organism:

S. pltaeochromogener NRRL B2119 0. 3 0.2 1. 4 S. griscomber 0.3 [1.2 4.5 S. purpeojuscus 1AM 0073.- 0. 3 0. 2 1. 3 S. oliaochremogmes A'lCC21,114 0. 4 0. 4 0. 2 0. 2 3. 4 S. olivoehromcpenea mutant CPO 3 1. 3 1.9 2. 2 2 3 6. 3

As these observations demonstrate, strains other than mutant strains CPC3 produced very little enzyme when cultured in all of the culture mediaemployed which did not contain xylose. However, the mutant producedenzyme in more copious quantities in the absence of xylose, and evengreater quantities when cultured in the presence of Xylose.

Characterization of the microorganism To characterize in greater detailthe microorganisms that are utilized in the practice of the presentinvention, the following information is presented. This information isassembled in a form that is similar to that used in describing theStreptomyccs genus of the Actinomycetales in Bergeys Manual ofDeterminative Microbiology, 7th Edition.

The first description applies to the parent strain, from which themutants were derived. The other descriptions apply to two mutantstrains, CPC 3 and CFO 15, that are preferred for use in the practice ofthe present invention. Strain CPC 15 is a single colony isolate ofstrain CPC 3.

Streptomyces olivochromogenes ATCC 21,114- (Parent) Aerial mycelium:Filaments with medium to close spirals.

Conidia ellipsoidal to spherical.

Gelatin stab: Good growth, Liquefaction within 10 days.

Agar: Wrinkled, tan to gray growth. Brown to brownishblack solublepigment produced.

Synthetic agar: White aerial and surface mycelium. No

pigment produced.

Starch agar: Abundant growth, yellow in color. Starch hydrolyzed.

Glucose agar: Abundant growth, tan to gray to dark gray in color.

Glucose broth: Thin, brown growth, flaky sediment.

Litmus milk: Dark brown ring; rapid coagulation.

Potato plugs: Abundant, white growth. Soluble brown to black pigmentproduced.

Nit'rites produced from nitrates.

Aerobic.

Grows well at 28-37 C.

Streptomyces olivochromogenes, mutant CPC 3 Aerial mycelium: Filamentswith medium to close spirals.

Conidia ellipsoidal to spherical.

Gelatin stab: Poor growth at 30 days; no liquefaction apparent.

Streptomyces olivochromogenes, mutant CPC l5 black soluble pigment Thefollowing mutant strains have been deposited at the American TypeCulture Collection and are being maintained there pursuant to a contractbetween that collection and the assignee of this patent application.

TABLE =8.STRAINS DEPOSITED Mutant strain No.: Culture deposit No.

CPC 3 21,713 CPC 4 21,714 CPC 15 21,715

The American Type Culture Collection has the following address: AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852.

The contract with the Culture Collection provides for permanentavailabilit of the culture to the public, upon issuance of the patent.The assignee of the present application has agreed that, if any of thesecultures on deposit should die, or is-destroyed, during the effectivelife of the patent, it will be replaced with a living culture of thesame organism.

Characterization of the enzyme A determination of the Michaelisconstants (Km) for reaction on xylose and on dextrose was made.

The purpose was to reveal the relative aflinities of the enzymepreparation for these substrates. At present, all isomerases that wehave examined that convert dextrose directly to levulose are xyloseisomerases. The determination was made utilizing sonic extract of theculture.

It was found that a lower Km Was obtained when the enzyme preparationacted on xylose than when it acted on dextrose. This established xyloseas the natural substrate of the isomerase, and the enzyme as a truexylose isomerase.

The capacity of the xylose isomerases of the mutants of the presentinvention to accept dextrose as a substrate is believed to be due toclose structural similarities between xylose and dextrose. Since themutants of the present invention produce the isomerase enzyme either inthe presence of xylose or in its absence, the enzyme is constitutiverather than necessarily induced only by the presence of xylose in theculture medium. When the culture medium does contain Xylose, the enzymemay be both constitutive and induced.

The following examples are presented to describe the invention further.

EXAMPLE 1 Enzyme production by mutant strain CPC 3 This exampledescribes the production of isomerase in accordance with one preferredmode of practicing the present invention.

Spores from a slant of mutant strain CPC 3 were inoculated into a 500ml. Erlenmeyer flask containing 100 ml. of a sterile medium composed ofthe ingredients described below in Table 9.

TABLE 9.INOCULUM MEDIUM COMPOSITION* Ingredients: Weight percent 15 D.E.corn syrup solids 2.0 Corn steep liquor (55% solids) 3.6 Magnesiumsulfate (MgSO -7H O) 0.05 Distilled water Balance *The medium is anaqueous solution with all weights caleulated as a percentage of thetotal medium, including water.

The pH of the culture medium was adjusted to about 7.1 with sodiumhydroxide and sterilized at 121 C. for 30 minutes. The flask wasinoculated and then incubated for about 60 hours at a temperature ofabout 28 C. on a reciprocating shaker at 120 cycles per minute.

For the second stage of development, a quantity of about 200 ml. ofsterile inoculum medium, having the composition described in Table 9,was prepared in each of several 1000 ml. Hinton modified Erlenmeyerflasks. A 10 ml. postion was then removed from the 500 ml. Erlenmeyerflask and transferred into the Hinton modified Erlenmeyer flask. Theinoculated flask was then agitated on a Gump rotary shaker at 224 cyclesper minute and incubated at a temperature of about 28 C., for about 48hours.

In the third step of development, four liters of the inoculum mediumdescribed in Table 9 was placed in a 7% liter bench fermentor andsterilized for 30 minutes at about 121 C. The bench fermentor wasinoculated from the one liter shake flask, and sparged with air at 4.0standard liters per minute. The fermentor was equipped with four bafllesand an agitator with two impellers of 3.0 inches diameter each. Theagitator was operated at a speed of 500 rpm. The fermentation wasconducted at about 28 C. for 48 hours.

Finally, 30 liters of culture medium having the composition described inTable 10, below, was placed in a 40 liter pilot plant fermentor andsterilized for 30 minutes at about 121 C.

10 TABLE 10.FERMENTATION MEDIUM Ingredients: Weight percent Corn steepliquor (55% solids) 3.6 Xylose 1.0 15 D.E. corn syrup solids 2.0 Glycine0.1 NH NO 0.2 MgSO -7H O 0.05

The pilot plant fermentor was then inoculated from the bench fermentor,and continuously sparged with air at the rate of 24.0 standard litersper minute, under a pressure of 15.0 p.s.i.g. The pilot plant fermentorwas equipped with four baflles and the agitator was equipped with twoimpellers. Each impeller had a blade diameter size of 4.5 inches. Theagitator was operated at a speed of 485 rpm. The fermentation wasconducted at about 28 C. for 48 hours.

After completion of the fermentation, the isomerase activity of theproduct broth was measured and found to be between 10 and 11 units permilliliter.

The enzyme production process was then repeated, but the medium in the40 liter fermentor was modified to contain starch rather than 15 D.E.corn syrup solids, the starch being present in the same amount as thecorn syrup solids it replaced. This medium produced enzyme at a somewhatslower rate, because the organism had to hydrolyze the starch. However,the end results indicated that no particular advantage in the use ofstarch in place of the corn syrup solids, so war as enzyme productionwas concerned.

EXAMPLE 2 Enzyme production by mutant strain CPC 4 Enzyme production:Variation in the culture medium and other operating parameters Severaladditional fermentations were conducted to produce culture broth havingisomerase activity.

In several of these fermentations, the culture medium was modified,generally at the 7 /2 liter and 40 liter stages. For the purpose of thisset of fermentations, a fermentation medium was prepared having thefollowing composition.

TABLE 11.FERMENTATION MEDIUM COMPOSITION Weight percent Ingredients:Medium B Corn steep liquor 4.0 Xylose 1.0 Glycine 0.1 Ammonium nitrate0.2 Magnesium sulfate heptahydrate 0.05 Dextrose 0.2 Starch 2.0

Variations were made in the fermentation media for differentfermentation runs, by the addition of small amounts of such additives asmore com steep liquor, 15 D.E. corn syrup solids, and the like.

All of the fermentations successfully produced culture broth containinga satisfactory level of isomerase activity, during fermentations of thetwo mutant strains, CPC 3 and CPC 4. Neither strain required thepresenceof cobalt in the medium to enhance enzyme production, and bothstrain'sproduced glucose isomerase in much greater quantities than theparent strain. Enzyme activities as high as 15 units per millilitterwere obtained, which is substantially higher than the activity generallyobtained upon fermentation with the parent strain.

EXAMPLE 4 Enzymatic isomerization To demonstrate the effectiveness ofthe isomerase produced by the mutant strains for converting dextrose tolevulose, several conversions were conducted. The enzyme preparationemployed consisted of frozen whole cells of either mutant strain CPC 3or mutant strain CPC 4. There do not appear to be any detectabledifierences in these enzyme preparations.

A series of conversions of 95 D.E. corn starch hydrolyzate wasconducted, using several different enzyme dosages. The conversions wereconduted at 70 C., at a pH of 6.25. Magnesium sulfate was added to thehydolyzate at the level of 0.01 molar. The dry substance level of thehydrolyzate was at about 600 mg./rnl. During isomerization, thehydrolyzate was maintained under an atmosphere of nitrogen, and the pHwas maintained by titration as necessary. The hydrolyzate was agitatedthroughout by stirring with bars in the conversion reactor, driven by amagnetic stirring motor.

The results are reported below in Table 12.

TABLE 12.DOSAGE SERIES WITHOUT COBALT Age of conversion in hours Percentconversion of 95 D.E. hydrolyzate to ketose Dosage u.]g.

s. 17. 7 so. 2 21. 9 35. 6 24. 7 37. 26. 0 40. l. 28. 1 41. 0 33. 4 43.2

TABLE 13.- CONVERSION AT 65 C. WITHOUT COBALT Age of conversion in hoursDosage, Percent of 95 D.E. hydrolyzate pH u.{g converted to ketose Theresults observed appearto demonstrate that the use of the higherconversion temperature of 70 C. is somewhat preferable. The higher pHlevels did not appear to produce any advantage.

- To summarize, it would appear that conversions conducted at 70 C., ata pH of about 6.25, and at a dry substance level in the range from about600 mg./m'l. to about 800 mg./ml., with an enzyme dosage of about1.2-1.4 units per gram, for from 40 to 48 hours approximately, and inthe presence of about 0.01 molar magnesium, in the absence of cobalt andwith nitrogen sparging, will produce highly satisfactory sweet syrupproducts. Compositions of the following kind can be expected:

TABLE 14.SWEET SYRUP COMPOSITIONS Weight percent,

Ingredient: dry substance basis Levulose 40-44 Dextrose 45-50 Highersaccharides 7-8 These data demonstrate that a 40% levulose syrup can bemade economically from a D.E. hydrolyzate utilizing enzyme preparationsderived from the mutants of the present invention, without the use ofadded cobalt.

Conclusion Although the taxonomy-of several strains of the mutantmicroorganisms of the present invention has been reported above, withsome attention to morphology, it will be obvious to those skilled in theart that the production of a physiological mutant may be accompanied bymorphological changes, but that the variation in biochemical activity isnot related to a specific morphological change. It will also be obviousthat a particular mutant may be isolated from natural sources as well asfrom survivors of exposure to artificial mutagenic agents.

Moreover, it will also be obvious that within the Streptomyces genus,the same type of mutant can be obtained from different species of xyloseisomerase producers, and that the type of mutant produced upon exposureto mutagenic agents is not dependent on the type of mutagenic agentused.

Therefore, although descriptions of the taxonomy of one parent strainand of two mutant strains, derived from the parent with ultravioletirradiation, have been reported in detail above, the descriptions of themutants do not necessarily characterize all strains, variants, orsub-mutants 0f the new mutants, nor do they necessarily distinguish thenew mutant forms from other strains of Streptomyces olivochromogenes.

Mutant strains of microorganisms that can be employed in the practice ofthe present invention can, in general, be readily identified byapplication of the following criteria:

l. Taxonomy that is characteristic of the Streptomyces genus.

2. Production under identical conditions of cultivation, particularlyunder those conditions described herein, of at least 50% more xyloseisomerase activity than Streptomyces olivochromogenes 21,114, andpreferably, twice as much. 7

3. Production of appreciable quantities of xylose isom erase whencultivated in nutrient media free of xylose and xylose-supplyingmaterials.

4. Preferably, in addition, production of appreciable quantities ofxylose isomerase when cultivated in nutrient media free of added cobalt.

The particularly preferred mutant strains are those of S.olivochromogenes.

The terms, a Streptomyces mutant, and, a mutant strain of S.olivochromogenes, and the like, as used in this application, areintended to include those cultures of microorganisms of the Streptomycesgenus that are identifiable by the above criteria. These terms thereforeinclude naturally occurring variants and artificially induced variantsof strains that are specifically characterized herein and that are alsoidentifiable by the above criteria.

The enzyme preparation that is produced from the mutant strains ofmicroorganisms that can be used in practicing the present invention maytake substantially any desired form. Good isomerization results havebeen observed when alcohol dehydrated cells were employed for effectingthe conversion, at a dosage of about 1.3 units of activity per gram ofdry substance. For example, when a 30 B. corn starch hydrolyzate at 95D.E. was con- 13 verted at an enzyme dosage of 1.3 units of activity pergram of dry substance at 70 C. (158 F.), utilizing alcohol dehydratedcells, at pH 6.25, for 46 hours, a levulose content of 40% was achievedin 37 hours and the final levulose content of the product was about 41%.The sweet syrup product filtered easily and refined well.

Other forms of enzyme preparation can also be used effectively. Onepreferred form of enzyme preparation is the culture broth that isremoved from a fermentor on a continuous basis during a continuousfermentation, since this represents a very economical productiontechnique.

Enzyme preparations obtained in accordance with the present inventiongenerally will be used to isomerize dextrose at a pH in the range fromabout 6 to about 7, and at a temperature in the range from about 60 C.to about 70 C. However, they are operative to effect isomerizationoutside of these ranges.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodification, and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice in the artto which the invention pertains and as may be applied to the essentialfeatures hereinbefore set forth, and as fall within the scope of theappended claims.

What is claimed is:

1. A process for the production of levulose comprising:

(a) subjecting a microorganism of the Streptomyces genus to the actionof a mutagenic agent;

(b) isolating a mutant strain of the microorganism that has the capacityto form an appreciable quantity of xylose isomerase when cultivated in anutrient medium that is free of xylose and xylose-supplying materials;

(c) cultivating the isolated mutant strain in a nutrient medium toproduce xylose isomerase; and

(d) isomerizing dextrose to levulose with said xylose isomerase.

2. A process in accordance with claim 1, wherein the microorganism is S.olivochromogenes.

3. A process in accordance with claim 2, wherein the isolated mutantstrain is selected from the group consisting of S. olivachromogenes ATCCNo. 21,713, S. olivochromogenes ATCC No. 21,714 and S. olivochromogenesATCC No. 21,715.

4. The process of claim 1, wherein said isolated mutant strain iscultivated in a medium containing xylose or a xylose-supplying material,to thereby produce a greater quantity of xylose isomerase than untreatedmicroorganisms cultured under the same conditions.

5. The process of claim 1, wherein said isolated mutant strain isfurther characterized by its capacity to form an appreciable quantity ofxylose isomerase when cultivated in a nutrient medium that is also freeof added cobalt.

6. The process of claim 1, wherein said xylose isomerase is in itssoluble form.

7. A process for the isomerization of dextrose to levulose, with axylose isomerase enzyme preparation that comprises subjecting a solutioncontaining dextrose to the action of a xylose isomerase enzymepreparation, said xylose isomerase enzyme preparation having beenprepared by cultivating, in a nutrient medium, an isolated mutant strainof a microorganism of the Streptomyces genus, said mutant strainproduced by subjecting said microorganism to the action of a mutagenicagent, said isolated strain being capable of reproducing itself to astrain having the characteristics of its parent mutant strain, saidmutant strain being further characterized by its capacity to form anappreciable quantity of xylose isomerase when cultivated in a nutrientmedium that is free from xylose and xylose-supplying material.

8. The process of claim 7, wherein said solution containing dextrose isa starch hydrolysate having a D.E. of at least about 95, said solutionbeing maintained at a temperature of from about C. to about C. and a pHof from about 6 to about 7.

References Cited UNITED STATES PATENTS 3,654,080 4/1972 Bengtson et al19531 F 3,622,463 11/1971 Iizuka et a1 19531 F 3,625,828 12/ 1971Brownewell l-31 FOREIGN PATENTS 483,704 11/1966 Japan.

OTHER REFERENCES Lamanna et al.: Basic Bacteriology, 3rd ed., TheWilliams and Wilkons, C. 0., Baltimore, pp. 723-27 (1965).

A. LOUIS MONACELL, Primary Examiner T. G. WISEMAN, Assistant ExaminerUS. Cl. X.R. 66, 79, 102, 112

